Pseudomonas fluorescens-based biosensor for bioavailability analysis of CuSO4 and CuO nanoparticles (nano-CuO) in seventeen different ecotoxicological and microbiologial test media. In parallel, free Cu in these test media was analysed using Cu-ion selective electrode (Cu-ISE). In the case of CuSO4, both free and bioavailable Cu decreased greatly with increasing concentration of organics and phosphates in the tested media. A good correlation between free and bioavailable Cu was observed  indicating that the free Cu content in biological test media may be a reasonably good predictor for the toxicity of CuSO4. As a proof, it was demonstrated that when eleven EC50 values for CuSO4 from different organisms in different test media were normalized for the free Cu in these media, the difference in these EC50 values was decreased from 4 to 1.8 orders of magnitude. Thus, toxicity of CuSO4 to these organisms was attributed to the properties of the test media rather than to inherent differences in sensitivity between the test organisms. Differently from CuSO4, the amount of free and bioavailable Cu in nano-CuO spiked media was not significantly correlated with the concentration of organics in the test media. Thus, the speciation of nano-CuO in toxicological test systems was not only determined by the complexation of Cu ions but also by differential dissolution of nano-CuO in different test conditions leading to a new speciation equilibrium. In addition, a substantial fraction of nano-CuO that was not detectable by Cu-ISE  was bioavailable to Cu-biosensor bacteria. Thus, in environmental hazard analysis of (nano) particulate materials, biosensor analysis may be more informative than other analytical techniques. Our results demonstrate that bacterial Cu-biosensors either in combination with other analytical/speciation techniques or on their own, may serve as a rapid (eco)toxicological screening method.The analysis of (bio)available copper in complex environmental settings, including biological test media, is a challenging task. In this study, we demonstrated the potential of a recombinant  Thus, metal speciation in different environmental matrices  has received remarkable attention  and the −11 M [−6 M, i.e., 63.5 μg Cu/L) to be applied for environmental samples. In addition, interference of ISEs with metal-organics complexes and non-target inorganic ions  has been discussed [Despite the importance of heavy metal speciation in interpretation of the results of toxicological assays, the available techniques to determine metal speciation and bioavailability are relatively limited. One of the most robust and probably the most widely applied techniques for speciation analysis is the ion-selective electrode (ISE) ,7. Unfor−11 M ), the deiscussed ,10. Despiscussed . On the iscussed ,14, whiciscussed . These miscussed ,17 and miscussed ,19.4 and nano-CuO) determined using a Cu ion-selective electrode (Cu-ISE) and a Cu-specific bacterial biosensor. Seventeen different standard ecotoxicological and microbiological test media were analysed with the aim to: (i) determine the complexing potential of these media for CuSO4 and nano-CuO and (ii) compare the responses of the Cu-biosensor and Cu-ISE. In parallel, Cu speciation was calculated using the Visual MINTEQ equilibrium model. The results for Cu speciation in the selected test media were used to calculate the amount of free ions at reported experimentally determined toxicity values (E(I)C50) for different aquatic test organisms.In this study, we performed a comprehensive comparison between free and bioavailable copper  was purchased from Riedel-de-Haën. 63.5 g Cu/L stock solution was prepared in deionised (DI) water and stored in the dark at room temperature. CuSO4·5H2O was added to the test media as 100-fold concentrated stock solution in water.CuSOet al. [Nano-CuO (advertised particle size 30 nm) was purchased from Sigma-Aldrich. The primary size of nano-CuO was confirmed to be 31 ±12.8 nm in an earlier study by Blinova et al. . TEM andet al. ,21. 63.5et al. . Hydrody2.2.The list of standard ecotoxicological and microbiological media used in this study is presented in Concentration of Cu in the test media was also determined by AAS-graphite furnace method according to the standard procedures (EVS-EN ISO/IEC 17025:2005) in a certified laboratory of the Institute of Chemistry, Tallinn University of Technology (Estonia). Additionally, pH (Orion PerpHect ROSS) and conductivity  of the test media were measured. Conductivity of DI water was measured to be 0.0003 mS/cm.2.3.2SO4, followed by polishing of the sensor surface with Al2O3 polishing strip. Electrode was calibrated daily using 10−8 M (6.4 × 10−4 mg Cu/L)–10−1 M  CuSO4·5H2O dilutions in DI water. Prior measurement, the ionic strength of all solutions was adjusted by supplementing the sample with 0.1 M NaNO3. Ionic strength of the test solution was adjusted because of its importance in the response of ISE as shown by Sauvé et al. [4 and nano-CuO were prepared in 5 mL of test media and the measurements were conducted in 30 mL polypropylene tubes. Dilutions of nano-CuO were allowed equilibrate for 2 h at 30 °C before the measurement. The limit of detection (LOD) of Cu-ISE in each test media was calculated as recommended for ion-selective electrodes by IUPAC [LOD)  was used. Before measurement, the electrode was thoroughly washed with DI water, then with 0.025 M Hé et al. . Approprby IUPAC . BrieflyAC [LOD) . The LOD50 of different organisms for CuSO4 and nano-CuO. The EC50 values were obtained from earlier published studies . Experimental EC50 values were obtained in exactly the same media (composition of the media was verified) that were used for Cu speciation analysis in this study , and cells were separated by centrifugation at 5,000 ×g for 10 min. Cell pellet was washed twice with 20 mL of appropriate test medium and further diluted with the same medium until OD600 ∼ 0.1 . 100 μL of CuSO4 or nano-CuO dilution or medium only (blank medium control) was pipetted onto white polypropylene 96-well microplate ; 100 μL of bacterial suspension was added to each well and plates were incubated at 30 °C for 2 h. Bioluminescence was measured using Orion II luminometer  and response of sensor bacteria to copper compounds was calculated as follows:The Gram-negative Cu-sensing  Cu ions , was useTwo parallel experiments were included per individual assay and three independent individual assays were performed. Due to the different potency of the cells to induce bioluminescence in ‘nutritionally’ different media absolute induction values in these media differed. Thus, the induction of the bacterial biosensor was expressed as %, where maximum induction value was considered as 100 and induction in blank medium as 1. Limit of the detection (LOD) of the Cu-biosensors was set at 20% induction .3.3.1.Vibrio fischeri), algal medium (used for the toxicity testing with Pseudokirchneriella subcapitata according to OECD 201), two artificial freshwaters . In addition, common media used for cultivation of microorganisms were included: LB medium (an undefined rich medium that supports the growth of variety of bacteria), malt extract (ME) and YPD (Yeast Peptone Dextrose medium) both used for the cultivation of yeasts  and bioavailability of copper in seventeen different media. The media selection included various standard ecotoxicological test media: 2% NaCl    have been relatively widely applied to study the speciation of e.g., certain heavy metals in different environmental conditions  and the  in LOD)  was loweCu ions) , Table 2appeared . Howeverum model . At the ectively . The preectively .4 in organics-containing media (LOD. Addition of 0.5% (wt) of AA to 0.9% NaCl increased the Cu-ISELOD by 87-fold  complexes were also bioavailable to Cu sensor bacteria [Contrary to Cu-ISE, which is a well established method for metal speciation analysis, bacterial metal-specific biosensors are currently largely in their developmental stage. Indeed, despite their more than 20-year history, they have not been widely applied yet for real environmental analysis . Most libacteria . Due to 4 in different media, one must note that being a live bacterial cell, this recombinant biosensor is not ‘operating’ at extremely low ionic strength solutions . The latter usually results in a relative poor bioluminescent response of sensor bacteria to copper if analysed in low organics-containing media: the bioluminescence in live bacteria consumes considerable amounts of cellular energy [Before discussing the response of the bacterial biosensor to CuSOr energy  and in eP. fluorescens OS8::KnCueRPcopAlux bacterial biosensor and Cu-ISE in CuSO4 spiked media were significantly correlated: the corresponding r value was 0.854  and nan mg Cu/L ) were alst media . Althougn et al.  demonstr [et al.  where th [et al. . One reaobtained . Thus, iobtained .Overall, there was a good correlation between LODs of Cu-ISE and Cu-biosensor bacteria for nano-CuO  of Cu2+ for fish from the literature were compared. Thirteen EC50 values for zebrafish, rainbow trout, trout, common carp, gibel carp and mullet were included; these values ranged for almost two orders of magnitudes from 0.03 till 1.4 mg/L, the median value being 0.21 mg/L. According to [Usually, the difference in toxicity of copper to several freshwater and saltwater organisms varies remarkably, ranging from 0.005 to 10 mg/L . When a rding to , this va50 values) for CuSO4 and nano-CuO which were determined using standard ecotoxicological test organisms  and yeasts . This brings us to the conclusion that the apparently big differences observed in toxicity of CuSO4 towards these organisms could be attributed to the properties of the testing media rather than to inherent differences in sensitivity between the test organisms. Unfortunately, we were unable to calculate the bioavailable Cu corresponding to respective EC50 values using the Cu-biosensor bacteria as all the EC50 values for CuSO4 exhibited already strong toxic effects towards the Cu-biosensor  enhanced dissolution of copper due to the increased dispersion of CuO and (ii) complexing of dissolved Cu by organics present in the medium. Overall, the free Cu measured with the Cu-ISE and bioavailable Cu measured by the Cu-biosensor correlated well. Interestingly, in nano-CuO suspensions, more bioavailable Cu  than free Cu (ISE) was detected. Thus, we suggest that bacterial biosensors were able to access additional fraction of nano CuO that was not dissolved and detectable by Cu-ISE. Hence, in the environmental hazard analysis of metal-containing (nano) particulate materials, biosensor analysis may be more informative than other respective analytical techniques. We also demonstrated that the remarkable difference in sensitivity of various aquatic organisms towards copper may be largely explained by the differential speciation of this metal in the test media used. Therefore, although very different aquatic organisms were compared  the concentrations of free copper harmful to these organisms were quite similar. This suggests that there seems to be no big inherent differences in sensitivity towards copper between different types of organisms. Moreover, the effect of composition of the test media should be considered as one of the most important factors in interpreting the results of toxicity tests.In this study, we used a Cu ion-selective electrode (Cu-ISE) and a bacterial Cu-biosensor in parallel, to analyze the speciation of CuSO


To explore the functional consequences of cannabinoid withdrawal in the rat mesolimbic dopamine system, we investigated the anatomical morphology of the mesencephalic, presumed dopaminergic, neurons and their main post-synaptic target in the Nucleus Accumbens. We found that TH-positive neurons shrink and Golgi-stained medium spiny neurons loose dendritic spines in withdrawal rats after chronic cannabinoids administration. Similar results were observed after administration of the cannabinoid antagonist rimonabant to drug-naïve rats supporting a role for endocannabinoids in neurogenesis, axonal growth and synaptogenesis. This evidence supports the tenet that withdrawal from addictive compounds alters functioning of the mesolimbic system. The data add to a growing body of work which indicates a hypodopaminergic state as a distinctive feature of the “addicted brain”. Thesant (SR) -7, thereant (SR) , 9.The morphological analysis of neurons , 11 has 9 TetraHydroCannabinol (THC) treatment has been shown to increase the length of the dendrites as well as the number of dendritic branches in both the shell of the NAcc and the medial prefrontal cortex, though not in other brain areas [Cannabinoid withdrawal produces a marked reduction of electrophysiological activity in NAcc-projecting dopamine (DA) containing neurons of the rat midbrain  and a rein areas . HoweverThus, in the present study we investigated the morphological alterations of Ventral Tegmental area (VTA) and Substantia Nigra pars compacta (SNc) DA neurons and their accumbal post-synaptic counterparts, the spiny neurons (MSN). TH-positive neurons and Golgi-Cox stained MSN were evaluated after chronic cannabinoid treatment and withdrawal in order to obtain further insights into the morphological features of the mesolimbic DA system and its involvement in cannabinoid dependence. In addition, the role of endogenous cannabinoids was investigated through administration of SR to drug-naïve rats.ad libitum. Experimental protocols were approved by the Ethical Committee of the University of Sassari and performed in strict accordance with the EC regulations for the use of experimental animals (CEE N°86/609), and recommended guidelines for the care and use of experimental animals approved by the Society for Neuroscience. Rats were administered twice daily (08:00 a.m. and 20:00 p.m.), for 6.5 days with either Δ9-THC  or CP 55,940  emulsified in 1% Tween 80, then diluted in a saline solution and administered i.p. in a volume of 3 ml/Kg.Male Sprague-Dawley albino rats , weighing 200-225 g at the beginning of treatment, were used. Rats were kept on a 12-h light/12-h dark cycle with food and water available Animals were assigned to the following groups: Chronic saline  (n=6); Chronic THC (15 mg/kg) (THC-C); 24 hrs spontaneous withdrawal from chronic THC (THC-W); precipitated withdrawal from chronic THC (15 mg/kg/administration) (THC-SR) (n=6); SR (5 mg/kg) in Sal (SR) (n=6) (6). On the morning of day 7 rats received the first daily administration  and 1 hour before sacrifice were tested for signs of spontaneous and SR precipitated-withdrawal. Animals were anaesthetized with urethane (1.3 g/kg i.p.) before transcardiac perfusion with 100 ml of ice-cold saline solution immediately followed by 400 ml of ice-cold 4% paraformaldehyde. Brains were divided into two parts at -2 mm from bregma approximately.The posterior part of brains, VTA- and SNc-containing, was post-fixed for 24 hours in the 4% paraformaldehyde solution and cryoprocteted in 30% sucrose in phosphate-buffered saline (PBS). Coronal sections between -5.80 mm and -6.30 mm (25 µm thick) from bregma, according to Paxinos and Watson  were obtAfter perfusion, the anterior part of the brains, NAcc-containing, were immediately rinsed (15 min x 3 times) in 0.1 M PBS and immersed in a Golgi-Cox solution. The solution was changed once after 2 days and the brains were then left in fresh Golgi–Cox solution for additional 14 days. After this period the brains were cryoprotected with a 30% sucrose solution for 2-3 days. 50 µm thick coronal slices, beginning at 1.70 mm and ending at 0.70 mm from bregma, according to Paxinos and Watson , were obMaximum intensity algorithm was used for 3D recostructions of TH-immunolabeled cells, while Extended focus algorithm was used for 3D recostructions of Golgi-Cox stained neurons (Bitplane Imaris V.7).Leica 4D Confocal Laser Scanning Microscope (CLSM) with an argon-krypton laser were used to analyse TH-positive neurons and Golgi-Cox stained sections. Confocal images were generated using 40x oil (na=1.00-0.5) and 100x oil (na=1.3). Each frame was acquired eight times and then averaged to obtain noise-free images. Optical sections, usually at consecutive intervals of 0.5 µm in Z axis, were imaged through the depth of the labelled neurons and saved as image stacks as previously described , 14. Max2).Morphometric analyses were performed by two independent observers blind to pharmacological treatments. TH-immunolabelled somata (80/group) were collected from a square area (approx. 200 µm/side). When totally included in the sections, TH-positive neurons were three-dimensionally reconstructed and used for measurements and statistical analysis using Bioscan Optimas software (version 6.5.1). The cell bodies were manually marked following their profile, excluding all dendritc trunks to measure their area (µmFor each treatment dendritic 80/group segments (at least 20 µm long) of second order dendrites were collected for analysis, from 0.7 to 1.70 mm from bregma (58) and identified by confocal rendered cells. Spines’ density was calculated by tracing a 10-15 µm long “sp” line, along the dendritic trunk and counting the number of spines therein. The procedure was repeated along the entire dendritic length from the bifurcation from the first branch of primary dendrites to the next bifurcation.Data was processed by a one-way ANOVA followed by post-hoc comparisons using the Bonferroni criterion.1) and in the dorsomedial portion of SNc [639=82.83; P<.0001), among experimental groups in the VTA. Post hoc analysis revealed that cell bodies in the VTA exhibited smaller somata under both THC withdrawals. In particular, a mean reduction was found for THC-W  and THC-SR , when compared with control. Surprisingly, we also found qualitatively similar changes in the SR group  whereas no changes were observed in THC-C group . No statistical differences were found for TH-positive neurons from SNc , . Confocal datasets of TH-immunolabeled neurons located in the VTA , but not in the core , on spine density in the experimental groups .One way ANOVA revealed a significant effect in the shell (F 158= 14.4 P<0.0001) and pharmacologically precipitated withdrawal (THC-SR t158= 12.6 P<0.0001) vs Sal. Interestingly, a reduction in spine density was also found in the SR group when compared to control . Further, post hoc analysis failed to reveal any significant difference between spine density counts in the shell MSN for THC-C  groups vs saline treated rats.Post hoc analysis, in the shell, showed a selective reduction in spine density for spontaneous THC-W . While further experiments are needed to corroborate this hypothesis, the present finding supports the idea of an endocannabinoid trophic and protective role , 26 at tIrrespective of the mechanisms underlying our observations, the structural changes occurring at both pre- and post-synaptic levels are likely to have profound consequences on dopaminergic transmission in the shell of the NAcc. Indeed, the reduced dopamine firing  is accomDendritic spines are particularly important in synaptic plasticity in reason of their rapid changes in volume and/or shape in response to stimuli. Is it true that spine number is very variable and they can spontaneously appear or disappear, but, what could then be the functional consequence of remodelling or losing about 30% of the dendritic spines? There are at least two mechanisms directly related to the reduction in the number of synapses, which can affect the overall firing rate of the neuron. The reduction of neuronal membrane associated with the loss of spine increases the input resistance of the neuron and, in principle, results in a more excitable neuron. On the other hand, the neuron may decrease its firing rate because the overall excitatory input is reduced.Overall, the present data suggest that the altered architecture of the DA system projecting to the shell of the NAcc documented here would profoundly alter the synaptic equilibrium affecting various neurotransmitters involved in the neurobiological mechanisms of cannabis dependence , 16.On the basis of the present and previous findings , 15, 18 In general, the present data lends further support to the notion that drug addiction can be seen as a chronic drug-induced, aberrant, form of neural plasticity , 16, 38,


Carbon nanotube (CNT) membranes allow the mimicking of natural ion channels for applications in drug delivery and chemical separation. Double-walled carbon nanotube membranes were simply functionalized with dye in a single step instead of the previous two-step functionalization. Non-faradic electrochemical impedance spectra indicated that the functionalized gatekeeper by single-step modification can be actuated to mimic the protein channel under bias. This functional chemistry was proven by a highly efficient ion rectification, wherein the highest experimental rectification factor of ferricyanide was up to 14.4. One-step functionalization by electrooxidation of amine provides a simple and promising functionalization chemistry for the application of CNT membranes. Conformational change of proteins actuated by voltage can open or close the gate of the channel, which regulates ion permeation with high selectivity -4. It inreatment . Neutralreatment .−1 in methanol oxidation [To achieve gatekeeper activity on CNT membranes, there needs to be a high functional density only at the CNT tips or pore entrances ,21. Thisxidation .The primary hypothesis of this paper is that the efficiency of voltage gatekeeping can be enhanced to obtain high on/off ratio using electrooxidation of amine in one step. The conformational changes of tethered dye molecules under bias will be identified by non-faradic electrochemical impedance spectroscopy (EIS) measurements. The EIS spectra can prove the effectiveness of this single-step functionalization on double-walled carbon nanotube (DWCNT) membranes. Transmembrane ionic rectification will be measured to compare the efficiency of gatekeeping. Stronger rectification indicates more efficient gatekeeping. The gatekeeper density is still unknown in our previous work. This can be quantified by dye assay on glassy carbon due to its similar structure with CNTs. A single-step modification may give higher overall functional density over a complicated two-step modification.2) were glued over a 3-mm diameter hole in polycarbonate plate (1-mm thick) to act as mechanical support. The top of the membrane was referring to the surface in the recess of the hole in the polycarbonate support, while the bottom of the membrane was on the bottom plane of the polycarbonate support. Pd/Au (30 nm) was sputter-deposited on the bottom of the membrane to give electrical contact to the CNT membrane and to act as effective working electrode.DWCNTs with average inner diameter of 2 nm and length of 30 μm were purchased from Sigma-Aldrich Corporation  image as seen in Figure N′,N′-dimethylamino) propylcarbodiimide hydrochloride and 5 mg of N-hydroxysulfosuccinimide were dissolved into 4 ml of 50-mM Direct Blue 71 dye in 0.1 M 2-(N-morpholino) ethane sulfonic acid buffer for 12 h at ambient temperature.To avoid grafting in the inner core of CNTs, CNT membranes were placed in U-tube fittings under a 2-cm inner solution column pressure. In two-step functionalization, as-prepared DWCNT membranes were first modified by flow electrochemical grafting with 5-mM 4-carboxy phenyl diazonium tetrafluoroborate/0.1-M KCl solution at −0.6 V for 2 min. In the next step, Direct Blue 71 dye (Sigma-Aldrich) was coupled with the carboxyl group on the tip of CNTs with carbodiimide chemistry: 10 mg of ethyl- in the three-electrode cell. The CNT membrane, with sputtered Pd/Au film (approximately 30-nm thick) on the membrane's back side, was used as the working electrode; Pt wire was the counter electrode, and the reference electrode was Ag/AgCl. Before electrografting, the ethanol solution of 0.1 M LiClO2. Linear scan was from −0.60 to +0.60 V with the scan rate at 50 mV/s.The schematic of the ionic rectification setup is shown in Additional file Toluidine blue O was reported to quantify the carboxyl group density on the polymer film. Our dye assay method was similar to that of previous reports ,44. GlasThe fabrication of DWCNT membranes using microtome cutting method was described in the ‘Methods’ section. TEM image of DWCNTs and SEM image of the as-made DWCNT membrane in cross-sectional view are shown in Figure In order to compare the gatekeeping efficiency of two different functional chemistries, transmembrane ionic rectification was measured on DWCNT-dye membranes. Figure Rct) 2.3 times in 20 mM KCl  compared to single-walled CNTs SWCNTs, 1.4 nm), the rectification of small ion pairs  was not seen, as was for the case of SWCNTs . However.4 nm, thOn another modified membrane with one-step amine grafting, we compared the rectification factor of three different ions, namely ferricyanide, NDS, and sodium benzenesulfonate, to examine the role of anion size in being repelled by the modification of CNT tips. In Table Ferricyanide has a well-known redox potential of 0.17 V (vs. Ag/AgCl), and thus, an important control experiment was done to make sure that the observed rectification was not due to faradic current; instead, it was due to transmembrane ionic current. Cyclic voltammetry scans (−0.6 to 0.6 V) showed no redox reaction on both as-made and one-step functionalized DWCNT membranes in 50-mM ferricyanide . This resulted in a carbodiimide coupling efficiency of 18% on glassy carbon. The net sulfonate density for the one- and two-step reactions is both comparable at 0.9 × 1015 charges/cm2, where the less efficient electrochemical oxidation of amine is similar to the loss in efficiency for the carbodiimide coupling reaction. However, in the case of the DWCNT membranes, the two-step modification was not effective at showing rectification (Table The non-faradic (EIS) spectra indicated that the functionalized gatekeeper by a single step can be actuated to mimic the protein channel under bias. This functional chemistry was proven to be highly effective on the enhancement of ion rectification. The disappearance of rectification also supported its effectiveness after removing the grafted gatekeeper by plasma etching. Interestingly, no apparent change of rectification was seen for the two-step functionalization. The likely reason is that highly efficient functional density can be obtained by electrografting of amine in one step since the poor yield in the second step (carbodiimide coupling reaction) resulted in a significantly lower gatekeeper density on CNT membranes. To address this question, two- and one-step functionalizations were quantified using dye assay on glassy carbon due to its well-defined area and similar chemical reactivity to CNTs. The schematic mechanism of dye assay's absorption and desorption is shown in Figure e report . Compari14 molecules/cm2 on glassy carbon by dye assay, the same as that of two-step functionalization. However, no apparent change of rectification was observed for two-step functionalization. The dye molecules on the membrane by single-step functionalization are more responsive to the applied bias due to direct grafting on the conductive surface instead of the grafted organic layer. Another possible reason is that the actual yield of the second step of the two-step modification on CNT membranes may be much less than the calculated 18% yield on glassy carbon. One-step functionalization by electrooxidation of amine provides a simple and promising functionalization chemistry for the application of CNT membranes.DWCNT membranes were successfully functionalized with dye for ionic rectification by electrooxidation of amine in a single step. Non-faradic (EIS) spectra indicated that the functionalized gatekeeper by one-step modification can be actuated to mimic the protein channel under bias. This functional chemistry was proven to be highly effective on the enhancement of ion rectification, wherein the highest experimental rectification factor of ferricyanide was up to 14.4. The control experiments supported that the observed rectification was a result of transmembrane ionic current instead of electrochemical reaction of ferricyanide. With the decreasing size of ion, we have observed smaller rectification due to partially blocked ion channels. The rectification was decreased with the higher ionic concentration. It suggested that the rectification is attributed to both charge and steric effects at low concentration, while the steric effect is dominant at high concentration. After removing the dye, the DWCNT-dye membrane exhibited no enhancement of rectification. This control experiment supported that the rectification was induced by functionalized dye molecules. The saturated functionalized dye density by a single step was quantified at 2.25 × 10CNT: Carbon nanotube; EIS: Electrochemical impedance spectra; NDS: 2,6-naphthalenedisulfonic acid.The authors declare that they have no competing interests.XZ carried out the modification of CNT membranes, rectification measurements and drafted the manuscript. JW fabricated the CNT membranes. ZQC helped in technical support. BH supervised this study and revised the manuscript. All authors read and approve the final manuscript.3Fe(CN)6) solution. Linear scan from −0.60 to +0.60 V with the scan rate at 50 mV/s. Schematic rectification setup. Working electrode (W.E) is DWCNT membrane coated with 30-nm-thick Pd/Au film; reference/counter electrode (R.E/C.E) is Ag/AgCl electrode. Constant potential was provided using a Princeton Scientific Model 263A potentiostat. Both U-tube sides are filled with potassium ferricyanide , DWCNT-dye membrane; reference electrode (R.E), Ag/AgCl; counter electrode (R.E), Pt; AC magnitude, 10 mV; DC magnitude, −0.6, −0.3, 0, 0.3, 0.6 V; frequency, 100 kHz to 0.2 Hz. Platinum wire, Ag/AgCl, and DWCNT-dye membrane were used as counter, reference, and working electrodes. Click here for fileControl experiments on DWNT membrane to rule out redox current. Cyclic voltammetry scan on DWNT membrane from −0.6 to +0.6 V. Reference /counter electrode, Ag/AgCl; working electrode, DWNT membrane. Both sides filled with 50-mM potassium ferricyanide solution. No Redox peak is found on bare and modified DWNT membrane, which supports the current change that is from ionic rectification. Click here for fileControl experiments on glassy carbon to rule out redox current. (A) Cyclic voltammetry scan on glassy carbon in 2-mM ferricyanide solution and 2-mM ferricyanide solution with 0.5 M KCl. (B) Cyclic voltammetry scan on glassy carbon in 50-mM ferricyanide solution and 25-mM ferricyanide/ferricyanide solution . With the supporting electrolyte KCl, oxidation and reduction peaks were observed at 0.29 and 0.06 V, respectively. However, no redox peaks were found without KCl, which supports that no redox reaction occurred in the solution. Click here for file


In a simulation based on a pharmacokinetic model we demonstrated that increasing the erythropoiesis stimulating agents (ESAs) half-life or shortening their administration interval decreases hemoglobin variability. The benefit of reducing the administration interval was however lessened by the variability induced by more frequent dosage adjustments. The purpose of this study was to analyze the reticulocyte and hemoglobin kinetics and variability under different ESAs and administration intervals in a collective of chronic hemodialysis patients.The study was designed as an open-label, randomized, four-period cross-over investigation, including 30 patients under chronic hemodialysis at the regional hospital of Locarno (Switzerland) in February 2010 and lasting 2 years. Four subcutaneous treatment strategies  were compared with each other. The mean square successive difference of hemoglobin, reticulocyte count and ESAs dose was used to quantify variability. We distinguished a short- and a long-term variability based respectively on the weekly and monthly successive difference.No difference was found in the mean values of biological parameters  between the 4 strategies. ESAs type did not affect hemoglobin and reticulocyte variability, but C.E.R.A induced a more sustained reticulocytes response over time and increased the risk of hemoglobin overshooting . Shortening the administration interval lessened the amplitude of reticulocyte count fluctuations but resulted in more frequent ESAs dose adjustments and in amplified reticulocyte and hemoglobin variability. Q2W administration interval was however more favorable in terms of ESAs dose, allowing a 38% C.E.R.A. dose reduction, and no increase of Darbepoetin alfa.The reticulocyte dynamic was a more sensitive marker of time instability of the hemoglobin response under ESAs therapy. The ESAs administration interval had a greater impact on hemoglobin variability than the ESAs type. The more protracted reticulocyte response induced by C.E.R.A. could explain both, the observed higher risk of overshoot and the significant increase in efficacy when shortening its administration interval.NCT01666301ClinicalTrials.gov:  The implementation of erythropoiesis stimulating agents (ESAs) in clinical practice has been one of the most important innovations in the management of anemia in chronic hemodialysis (HD) patients. The ESAs therapy implicates however a non-physiologic stimulation of the erythropoietic process and has been identified as one of the most influential causative factors of hemoglobin variability in HD patients.Instability over time of the hemoglobin values is a physiological biological event, but is accentuated in HD patients under synthetic drug stimulation of the erythropoiesis . In factThe most recent history of ESAs has been signed by the development of long-acting compounds with distinct molecular structure compared with the original epoetin alfa and beta, such as Darbepoetin alfa  administration) ,7 and C.In the absence of evidence about the impact of the half-life and administration interval of ESAs on hemoglobin variability, we first performed a simulation based on a pharmacokinetic model. The task for the nephrologists participating in the study was to achieve and maintain, on a Visual Basic Excel table, the hemoglobin target using 3 different ESAs, with a half-life of 24, 48 and 138 hours, administered weekly or monthly. This study allowed us to demonstrate: (i) the hemoglobin variability decreases with an increase of the ESAs half-life and with a shortening of the administration interval; (ii) the monthly prescription compared to the weekly one is associated with less dosage adjustments, (iii) the number of ESAs posology modifications correlate directly with hemoglobin variability [Aware of the limits of the simulation tool, aiming at verifying our results in the clinical setting and in order to analyze the cellular kinetics of erythopoiesis under different therapeutic strategies, we studied the relationship between two relevant pharmacodynamic response parameters, hemoglobin and reticulocytes, under different ESAs  and administration intervals  in a collective of chronic HD patients.The study was designed as an open-label, randomized, four-period cross-over investigation Figure , consideWe enrolled 31 chronic patients aged 18 years or older, undergoing dialysis 3 times a week for at least 8 weeks before screening, necessitating continuous subcutaneous treatment with weekly Darbepoetin alfa or Erythropoietin beta to maintain hemoglobin (Hb) targets. Exclusion criteria were pregnancy or not respecting the above mentioned conditions. The patients were randomly assigned to subcutaneous C.E.R.A or Darbepoetin alfa, independent of previous ESAs therapy. Patients randomization was conducted by computerized procedure by a co-investigator; the randomization was balanced by groups of six. The randomization codes were communicated to the co-investigators implicated in ESAs prescription and to the dialysis nurses responsible of drug administration. Taking into account the administration of the study drug under supervision, no measure for monitoring compliance was indicated.Study drug was switched over at the beginning of each treatment period. Each period lasted 6 months; the first month of each period was not considered in the analysis (wash-in phase).The starting dose of ESA was based on patient’s cumulated dose in the month before randomization according to approved labeling .During the study, doses were adjusted to maintain Hb values within a range of 11.0-13.0 g/dl. Dose adjustments were scheduled at 4 weeks intervals (Q4W) during the first two periods (P1-2) and every 2 weeks (Q2W) during periods three and four (P3-4). P1-2 and P3-4 were intercalated by a 2-month transition phase, during which the drug study was switched but continued to be administrated at Q4W interval . A protocol directed ESA dose adjustment according to approved labeling [In the Hb target 11.0-13.0 g/dl, dose was continued unchanged. If the rise in Hb was greater than 2 g/dl in four weeks, dose was reduced by 25%. If the Hb exceeded 13.0 g/dl, dose was temporarily withheld until the Hb decreased under 12 g/dl, at which point therapy was reinitiated at 50% lower than the previous dose.If the increase in Hb was inadequate (less than 1 g/dl), the dose was further increased by 25%.When switching from Q4W to Q2W administration interval, the monthly ESA total dose was maintained unchanged (conversion factor 1:1), but divided in two administrations. Body weight was not integrated in the ESA dose adjustment protocol.Of note, Darbepoetin alfa is licensed in Switzerland for one-monthly administration, in contrast to the many other European countries .Supplementary iron carboxymaltose was administered if necessary to stabilize serum ferritin between 100 and 500 μg/l.Patients were assessed Q1W with hemoglobin values (Hb) and reticulocyte counts (ret), while serum ferritin was measured Q4W.Hemoglobin variability was the primary outcome while reticulocyte variability, induced reticulocyte response, risk of hemoglobin overshooting and ESAs efficacy comparing 4 to 2 week administration interval were the secondary ones.The results of the months following a red blood cells transfusion or an overt bleeding episode were not considered in the analysis. Data obtained during patient hospitalization for any reason were similarly suppressed.The protocol of the study was approved by the local Ethical Committee . All the patients gave written informed consent prior to enrolling in the study.To assess the time-variability of both hemoglobin and reticulocytes in Q4W, data were grouped over a four-week period corresponding to the number of weeks following each EPO injection. Linear mixed-effect models were applied on these data with individual random effects. A parabolic function appeared to best fit the data. Adjustments for age and sex as well as for the dose of EPO and iron were made. Reticulocytes were log-transformed to better approach normality. For Q2W, the time between two consecutive injections was too short to allow similar analyses to be performed.In absence of previous literature permitting an estimation of an expected between-groups difference, no sample size calculation had been driven. Moreover all patients who agreed to participate had been included in the study, so the sample size was the largest we could provide.The mean square successive difference (MSSD) was used to quantify temporal instability in terms of both variability and temporal dependency over time. The MSSD is the average of the squared difference between successive observations . We distBoth ST-MSSD and LT-MSSD were assessed for hemoglobin, reticulocytes and ESA (only LT-MSSD).Linear mixed-effect models predicting the effect of ESAs type on the variability (ST-MSSD and LT-MSSD) of hemoglobin were developed, and adjusted for ESAs administration order and ESAs administration interval.As ST-MSSD and LT-MDDS were right-skewed variables, log-transformed values were used in the analyses to better approach normality.Statistical analyses were performed using R (version 2.12.0) with the “lme” function from the “nlme” package . SignifiA total of 31 patients were enrolled in the trial and randomized into 2 treatment groups  at Q4W administration interval. Of these, 30 patients received at least one dose of the study drug, while 1 patient randomized to C.E.R.A died before the first study drug was administrated.During P1-2, 6 patients prematurely interrupted the assessment because of death  or renal transplantation .Out of the 24 patients who completed P1-2, 5 patients could not be included in P3-4, because of too low EPO dose, not allowing to be split in 2 half-doses. During P3-4, out of 19 patients , 4 patients prematurely interrupted the assessment because of death  or transfer to other hemodialysis centers .Patient demographic data, clinical characteristics and laboratory results at baseline are presented in Table The most frequent etiologies of chronic kidney disease (CKD) were hypertension (29%), miscellaneous (diabetes and hypertension) (25.8%), diabetes (6.5%), glomerulonephritis (9.7%), interstitial nephritis/pyelonephritis (6.4%) or other/unknown causes (22.6%).Data concerning hemoglobin, reticulocyte count, ferritin, cumulated ESAs and iron dose are presented in Table Overall, data of 572 patient-weeks under Darbepoetin alfa and 571 patient-weeks under C.E.R.A in P1-2, respectively 353 patient-weeks under Darbepoetin alfa and 359 patient-weeks under C.E.R.A in P3-4, were analyzed.With Q4W ESAs administration (P1-2) , suggesting any significant time course difference of hemoglobin response between ESAs. The hemoglobin level was slightly higher in participants under C.E.R.A than in those under Darbepoetin alfa, but the difference was not significant (p = 0.2). Similarly, the reticulocyte response followed an oscillatory cyclic movement over 1 month with both ESAs. The monthly oscillatory time course differed between C.E.R.A and Darbepoetin alfa (the interaction terms were clearly significant: p < 0.001 for time and p < 0.001 for time2). In participants under Darbepoetin alfa, the reticulocyte level reached its maximum within the first days after injection but it then decreased almost linearly, whereas under C.E.R.A, the maximum level was slightly lower, but it remained nearly constant up to two weeks after the injection (secondary outcome). We found a significant correlation between hemoglobin and reticulocyte count (p < 0.001) after adjusting for time, ESAs type, baseline Hb, ESAs-dose and cumulated dose, iron dose and cumulated iron.ation P1-   variability (ST-MSSD) and long-term (month-to-month) variability (LT-MSSD) are summarized in Tables For hemoglobin, no significant difference was found in ST-MSSD, when modifying ESAs or administration interval. However, a significant hemoglobin LT-MSSD increase was found when shortening the administration interval from Q4W to Q2W, both for C.E.R.A (p = 0.03) and Darbepoetin alfa (p = 0.02) (primary outcome). Reticulocyte LT-MSSD was found to predict hemoglobin LT-MSSD (p < 0.0001), after adjusting for ESA type, administration order and interval.Reticulocyte LT-MSSD increased when shortening the administration interval with both ESAs. The difference was significant in the Darbepoetin alfa group (p = 0.04), but didn’t reach significance in the C.E.R.A. group (p = 0.1).Reticulocyte ST-MSSD decreased significantly when shortening the administration interval from Q4W to Q2W, both for C.E.R.A (p = 0.01) and Darbepoetin alpha (p = 0.01).Concerning ESAs, when analyzing the start and final dose utilized in both study phases, we observed that: Darbepoetin alfa monthly dose significantly increased from mean (sd) 180 145) to 266 (172) μg over P1-2 (p = 0.002), without significant change over P3-4, from mean (sd) 202 (185) to 170 (168) μg (p = 0.3)  administered subcutaneously at different intervals on the cellular kinetics of erythropoiesis under conditions of standard clinical practice in hemodialysis patients and to verify the findings of a simulation based on a pharmacokinetic model suggesting a correlation between hemoglobin variability and both half-life and administration interval .Hemoglobin in dialysis patients is an instable parameter, influenced by several measure-related , patients-related , which implies the average of the squared difference between successive observations. Further, the MSSD allows to distinct short-term (e.g. week-to-week) from long-term instability, producing fluctuations across a long-term duration (e.g. month-to-month). MSSD could be a more precise estimator of hemoglobin instability over time.A second important limit of the studies about hemoglobin variability under ESAs is that they ignore the effect of ESAs on cellular production, namely the ESAs induced reticulocyte response. If and how reticulocytes are implicated in the phenomenon of hemoglobin variability is actually not known.With the current four-period cross-over study, we analyzed the influence of 2 ESAs  and 2 administration intervals  on hemoglobin and reticulocyte response. Therefore, 4 treatment strategies  were compared with each other.No difference was found in the mean values of biological parameters  between the 4 strategies. However, Q2W administration interval seemed to be more favorable in terms of ESAs dose, allowing over the 6 observation months a 38% C.E.R.A. dose reduction, and no increase of Darbepoetin alfa (secondary outcome). This finding about C.E.R.A. is concordant with previous observations . RetrospThe great sensibility of reticulocyte dynamic to ESAs therapy was evidenced by several factors, namely the evident oscillatory response induced by the ESAs pulse, the contracted short-term (week-to-week) variability when shortening the administration interval and splitting the ESAs dose, the amplified long-term (month-to-month) time instability under 2 week administration interval. Accordingly to its extended half-life, C.E.R.A. induced a more sustained erythropoietic response, as evidenced by the more protracted reticulocyte production under monthly administration intervals (secondary outcome). This can also explain the increased risk of hemoglobin overshooting (Hb > 12.0 g/dl) observed under C.E.R.A (secondary outcome).When compared to reticulocytes, the hemoglobin dynamic seemed to be less sensitive to ESAs therapy strategy. No significant difference was found in the oscillatory course using the two different ESAs. The administration interval impacted on the monthly Hb course (loss of the cyclic oscillatory movement under Q2W administration), without modifying short-term week-to-week variability. The long-term (month-to-month) variability increased when shortening the administration interval from 4 to 2 weeks, and this change was predicted by the increased reticulocyte long-term (month-to-month) variability (primary outcome).These results however, should be looked at carefully, taking into account the limitations of the study and the possible sources of bias.First, in absence of previous literature studies quantifying the impact of different ESAs treatment and administration protocols on hemoglobin temporal instability in terms of both variability and temporal dependency over time, we were not able to assume a degree of effectiveness of therapy allowing us to estimate the sample size. We therefore proposed the protocol to all patients under hemodialysis at our centre at study begin.Second, this is a randomized controlled study realized in a “real life” scenario. In order to assess the pharmacodynamic effect of ESAs on hemoglobin variability, an “on-treatment” analysis was performed, restricting the comparison of the treatments to the ideal patients-weeks, that is, those without intercurrent potentially perturbing event .Ideally, the four administration protocols should have been studied in a stable population, without intercurrent illness, but this is far away of being the reality of a hemodialysis station. A further characteristic of the dialysis population is its high instability , that explains the high drop-out rate (only 50% of the patients included reached the study end).In conclusion, reticulocytes seemed to be a more sensitive marker of time instability of the erythropoietic dynamic under ESAs therapy, their variability influencing the hemoglobin response. As expected, shortening the administration interval lessened the amplitude of reticulocyte count fluctuations. However, surprisingly enough, the same strategy was associated with an increased month-to-month reticulocyte and hemoglobin instability. This meaning, that in the clinical setting the favorable and unfavorable consequences of lengthening the administration interval on hemoglobin variability tend to counterbalance each other, contrarily to the results of our previous published pharmacokinetic computer-based simulation. However, even if these results are comforting, the non-physiologic and extreme reticulocyte count fluctuation over time using the monthly administration interval imposes prudence and suggests further exploring the possible unfavorable consequences on hemoglobin stability. Furthermore, the more protracted reticulocyte response induced by C.E.R.A.  could explain both, the observed higher risk of hemoglobin overshoot and the significant increase in efficacy when shortening from once to twice a month its administration interval.C.E.R.A: Continuous erythropoietin receptor activator; EPO: Erythropoietin; ESAs: Erythropoiesis stimulating agents; HD: Hemodialysis; Hb: Hemoglobin; Ret: Reticulocytes; ST-MSSD: Short-term mean square successive difference; LT-MSSD: Long-term mean square successive difference; Q4W: 4 Weeks interval; Q2W: 2 Weeks interval.The authors declare that they have no competing interests.VF and LG were involved in the study design, analysis and interpretation of the data and writing of the report; GB and IS participated in the sample collection, and interpretation of the data; AO and PV participated to the data analysis. MB helped formulate the study design, the data analysis strategy and contributed to the writing of the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/157/prepub


Obesity is an epidemic affecting over 1 in 3 adults in the United States. Patients who are obese have increased cardiovascular mortality compared to those with normal weight, partly due to direct effects on the heart. However, the effect of obesity on advanced measures of cardiac function such as strain, torsion and synchrony are poorly understood.Ten 12-week-old male C57BL/6 mice were randomized to one of two diets: 1) a high-fat ‘western' diet with 60% of calories from fat or 2) a low-fat diet with 10% of calories from fat. After 5 months on the diet, the mice were imaged on a 7T ClinScan MRI  using a cine DENSE protocol and a 4-element phased array cardiac coil. Three short-axis slices  and two long-axis slices (4-chamber and 2-chamber) were acquired with 15-20 frames per cardiac cycle. These data were then used to quantify left ventricular strain, torsion and measures of synchrony using semi-automated post-processing software written in MATLAB .Left ventricular sub-epicardial strain was significantly lower in the obese mice with a 40% reduction in circumferential strain (p<0.001), a 19% reduction in longitudinal strain (p=0.03), and a 53% reduction in radial strain (p=0.06). By contrast, left ventricular sub-endocardial strain was modestly reduced in the obese mice in the circumferential direction by 12% (p=0.01), and not significantly different in the radial (p=0.63) or longitudinal (p=0.44) directions. Peak left ventricular torsion was reduced by 34% in the obese mice (p=0.06). Finally, the radial uniformity of strain index showed a reduction in the synchrony of contraction in the left ventricle (p=0.01) with a time delay in the septal to free wall direction.Diet-induced obesity leads to a reduction in cardiac contractility in mouse models as evidenced by reductions in left ventricular strains and torsion. Reductions in cardiac strain are mostly limited to the sub-epicardial layer of the left ventricle, with relative preservation of function in the sub-endocardium. Diet-induced obesity also leads to reduced synchrony of contraction in the left ventricle of the heart.This work was supported by an NIH pilot project from a COBRE grant (5P20RR021954-05 and 8P20GM103527-05), the University of Kentucky Cardiovascular Research Center, and contributions made by local businesses and individuals through a partnership between Kentucky Children's Hospital and Children's Miracle network.


Patients on anti-tuberculosis treatment may develop acute kidney injury (AKI), but little is known about the renal outcome and prognostic factors, especially in an aging population. This study aimed to calculate the incidence of AKI due to anti-TB drugs and analyze the outcomes and predictors of renal recovery.From 2006 to 2010, patients on anti-TB treatment were identified and their medical records reviewed. Acute kidney injury was defined according to the criteria established by the AKI Network, while renal recovery was defined as a return of serum creatinine to baseline. Predictors of renal recovery were identified by Cox regression analysis.Ninety-nine out of 1394 (7.1%) patients on anti-TB treatment had AKI. Their median age was 68 years and there was male predominance. Sixty (61%) developed AKI within two months of anti-TB treatment, including 11 (11%) with a prior history of rifampin exposure. Thirty (30%) had co-morbid chronic kidney disease or end-stage renal disease. The median time of renal recovery was 39.6 days . Factors predicting renal recovery were the presence of fever, rash, and gastro-intestinal disturbance at the onset of AKI. Sixty-two of the 71 (87%) patients who recovered from AKI had successful re-introduction or continuation of rifampin.Renal function impairment is not a rare complication during anti-TB treatment in an elderly population. The presence of fever and rash may be associated with renal recovery. Rifampin can still be used in most patients who recover from AKI. Tuberculosis (TB) is a global disease affecting one-third of the world’s population . In 2011Reviewing literature of rifampin-induced AKI ,11-13, tThe present study was conducted to investigate the incidence of AKI during anti-TB treatment in Taiwan and to determine outcomes and predictive factors for renal recovery.This retrospective study was conducted at the National Taiwan University Hospital. The hospital’s Research Ethics Committee approved the study protocol. Patients were included if they met the following criteria: (1) age ≥18 years; (2) clinical diagnosis or suspicion of TB; (3) under rifampin-containing anti-TB treatment; and (4) had onset of AKI during anti-TB treatment. Acute kidney injury (AKI) was defined according to the criteria established by the Acute Kidney Injury Network (AKIN) and was classified into three stages (Stages 1 to 3) based on serial changes in serum creatinine level. Stage 1 was defined as an increase in serum creatinine ≥26.52 μmol/L or by 1.5-fold but less than twice the baseline level. Stage 2 was defined as a two-fold increase but less than three-fold increase from baseline, while Stage 3 was defined as a three-fold increase from the baseline level .Taiwan Guidelines for TB Diagnosis and Treatment. Inter-group difference was compared using the n of CKD .From 2006 to 2010, 2322 TB patients were identified, including 1394 with serum creatinine data before and after the start of anti-TB treatment. Of the 361 patients with increased serum creatinine level ≥17.68 μmol/L, 262 were excluded , and anemia (hemoglobin < 100 g/L) (Table p < 0.001) (Table The most common presenting symptoms at the onset of AKI were skin rash 21%) and gastro-intestinal disturbance (17%), followed by fever (6%) and arthralgia (4%)  patients Table . Among tPyrazinamide was discontinued in 50 (51%) patients after the onset of AKI. Anti-TB drugs were interrupted in 30 (30%), including seven (7%) who failed to complete the treatment . In the remaining 23 patients, the median duration of treatment interruption was 14 days .p = 0.002). Five of the six patients had hypoalbuminemia.Within a follow-up period of 180 days since the onset of AKI, two patients were lost. Twenty-six (27%) did not recover from AKI (AKI-unrecovered group), including six (6%)  who required long-term renal replacement therapy. The median baseline creatinine level of the six patients was 221 μmol/L , significantly higher than those of the other 93 patients (p = 0.005) and the prevalence of hematuria (p = 0.033) and proteinuria (p = 0.048) at the onset of AKI were significantly higher in the AKI-unrecovered group, whereas thrombocytopenia was less common (p = 0.064) (Tables p = 0.061) (Table Among the 71 patients who recovered from AKI (AKI-recovered group), 64 (90%) recovered within 100 days. Serum BUN level  Tables . The AKIIn the Kaplan-Meier analysis, fever (HR 3.65 [1.43-9.37]), gastro-intestinal disturbance (HR 2.32 [1.27-4.27]), and thrombocytopenia (HR 2.20 [1.08-4.50]) at the onset of AKI were significant predictors of renal recovery, whereas skin rash (HR 1.69 [0.99-2.90]) and arthralgia (HR 2.78 [0.99-7.78]) had borderline significance , whereas thrombocytopenia  and gastro-intestinal disturbance  had borderline significance.Sensitivity analysis focusing on the sub-population without CKD revealed that fever at AKI onset remained a significant predictor of renal recovery  , implying that the study subjects here are representative of the whole TB population in Taiwan . With thThe mechanism of rifampin-induced AKI is not well established. Several studies suggest that it is either a type II or type III hypersensitivity reaction induced by rifampin antigens in which anti-rifampin antibodies form immune complexes that are deposited in renal vessels, the glomerular endothelium, and the interstitial area . These rIn previous studies, more than 80% of patients recover from AKI within 120 days ,11-13. TThe prognostic factors of AKI during anti-TB treatment are rarely investigated. Only the duration of anuria and leukocytosis have been associated with renal recovery . The curMore than 50% of the AKI events occurred within two months of anti-TB treatment, indicating that an acute phase reaction may be contributory. The findings also suggest that patients with CKD and hypoalbuminemia maybe more vulnerable to severe and permanent renal damage. After AKI develops, more physicians decide to discontinue pyrazinamide, rather than rifampin, implying that they do not know which of the first-line anti-TB drugs is the most common offending drug for AKI. Continuous medical education on the correct regimen modification is necessary to prevent further renal damage in TB patients with AKI.In this study, the diagnosis of AKI is not confirmed because renal biopsy was not performed. However, the results of previous studies suggest that even without histology studies, the diagnosis of rifampin-induced AKI can be made based on the typical time course and by excluding other etiologies . In the Re-treatment or re-exposure to rifampin causes repeat antigen exposure, which can lead to a high antibody surge and subsequent severe immune response ,26. ThisThe present study has some limitations. First, there is no strong evidence to confirm rifampin as the cause of AKI due to the lack of pathology results. Only seven patients had a second AKI episode after re-challenge rifampin. However, this may not be a serious problem because possible causes other than anti-TB medication have been excluded and AKI due to first-line anti-TB drugs other than rifampin is rarely reported ,6. SeconAnti-tuberculosis drug-induced acute kidney injury is not rare in an aging population. It usually develops within two months of treatment and resolves within three months after onset. Although about 27% of patients with AKI will have permanent renal impairment, those who present with fever, rash, and GI disturbance at the onset of AKI have better renal recovery. Of the 73% of patients who had recovery of renal function, 87% successfully continued rifampicin or had rifampicin re-introduced.The study was approved by the Institutional Review Board of National Taiwan University Hospital (NTUH_IRB: 9561707008).Tuberculosis is a global disease affecting one-third of the world’s population. Acute kidney injury is not uncommon in patients receiving anti-tuberculosis drugs. Identifying risk and prognostic factors of acute kidney injury is important in the management of tuberculosis.The authors declare that they have no competing interests.CHC, YFC, VCW, JYW: study concept and design, analysis of data, and drafting of manuscript. CCS, CHL: statistical analysis and interpretation of data. CHC, JYW: interpretation of data and review of manuscript. LNL, CJY: review of manuscript. All of the authors have seen and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/23/prepub


P = 0.006) was observed in non-diabetic controls. The serum vaspin concentration was also significantly correlated with body mass index (BMI) , waist-hip ratio (WHR)  and homeostasis model assessment of insulin resistance (HOMA-IR)  in T2DM patients. In cohort analyses, it was found that lower serum vaspin  and adiponectin  levels at baseline were risk factors for new onset of T2DM at follow-up. The percentage of insulin treatment in T2DM patients was higher in the sub-group with lower serum vaspin level than that in the sub-group with higher vaspin level at follow-up . Our study indicates that low serum concentration of vaspin is a risk factor for the progression of T2DM.Vaspin is a novel adipocytokine that has potential insulin-sensitizing effects. The aim of this study is to explore the role of vaspin in the progression of type 2 diabetes mellitus (T2DM) in humans through a longitudinal process. This was a 2-year follow-up study that included 132 patients with T2DM and 170 non-diabetic subjects. The serum vaspin and adiponectin levels were determined with ELISA. Anthropometric measurements, circulating glucose, hemoglobin A1c, insulin level, liver function, kidney function, and lipid profile were measured for each participant. The new onset of T2DM was counted in non-diabetic subjects and the glycemic control was analyzed in T2DM patients at follow-up. At enrollment, the serum vaspin and adiponectin levels were lower in T2DM patients compared with non-diabetic subjects. Significant positive correlation between serum vaspin and HDL-C levels (r = 0.23,  Adipose tissue is not only an inert energy-storing tissue, but also an active endocrine organ Vaspin , a novel adipocytokine, was firstly identified in obese OLETF rats. It has been suggested that vaspin has potential insulin-sensitizing effects Hypothetically, just like adiponectin, low vaspin level might be concomitant with poor glycemic control in diabetic patients because of its impact on insulin sensitivity and glucose metabolism. To our knowledge, the role of vaspin in glycemic control in diabetic patients has not been addressed in previous studies. In this 2.0-year follow-up study, our aims were to examine 1) whether serum vaspin levels are associated with adiposity and insulin-resistance; 2) whether non-diabetic subjects with lower serum vaspin levels are more susceptible to developing T2DM; 3) whether lower vaspin levels are concomitant with poorer glycemic control in T2DM.All subjects were local residents of Han ethnicity in Shanghai and consecutively recruited in Department of Endocrinology, Xinhua hospital, Shanghai Jiaotong University School of Medicine and Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine from Oct 2009 to Mar 2010. T2DM was diagnosed according to 1999 WHO diagnostic criteria The study was approved by the Human Ethical Review Committees, Xinhua Hospital and Shanghai Tenth People's Hospital, and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects.Blood samples for biochemical analysis were collected after overnight fasting for at least 10 hours, and immediately frozen in aliquots at −80°C until analysis. FPG, 2h-PG, serum liver and renal function and lipid profile were measured by using standard laboratory techniques on a Hitachi 7104 Analyzer . HbA1c was determined by using high-performance liquid chromatography  method. Serum insulin concentrations were determined by commercially available radioimmunoassay in non-diabetic subjects and insulin-naive T2DM patients . Insulin sensitivity index: homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by the formula as reported before At re-examination after two years, anthropometric indices, FPG, 2h-PG, HbA1c and lipid profile were measured in all subjects. Drug uses and concomitant diseases were also recorded at the same time. During the 2-year follow-up, FPG and HbA1c had been checked in diabetic patients every three months at clinic, and hypoglycemic agents were prescribed by experienced endocrinologists. For diabetic patients, during the stable therapeutical period of two years, insulin or oral antidiabetics (OAD) treatments were recorded. For non-diabetic controls, the incidence of T2DM was investigated, and the glucose levels and other metabolic indices were also analyzed at follow-up.t test or covariance analysis for continuous variables and a χ2 test for categorical variables. Pearson correlation coefficients were used to assess the associations of continuous variables with vaspin. Multiple stepwise logistic regression analysis was used to detect the risk factors for development of T2DM, with adjustment for potential confounding factors. Results are presented as mean (standard deviation) for continuous variables and as percentage for categorical variables. The serum vaspin and adiponectin concentrations were adjusted for sex in all analyses because of the higher levels of these hormones in women Statistical analysis was performed using the Statistical Package for the Social Sciences . All datasets were examined for normal distribution using the Kolmogorov-Smirnov test. The traits that deviated significantly from normal distribution were approximately normalized by logarithmic transformation. Differences between groups were examined using an unpaired P = 0.025). The P value was 0.041 after adjusting for the confounding factors including gender, age and BMI. The median (interquartile range) level of vaspin was 0.425 ng/mL  in non-diabetic group, and 0.353 ng/mL  in T2DM group. Higher adiponectin  was found in non-diabetic controls when compared with T2DM group. The P value was not significant after adjusting for the confounding factors including gender, age and BMI. The serum fasting insulin level was significantly higher in T2DM group than that in control group (P<0.05). In females with or without diabetes, both the serum vaspin and adiponectin levels were significantly higher than those in males . Detailed baseline clinical characteristics of all subjects were presented in P = 0.025) and lower percentage of smokers (7.2% vs. 22.0% P = 0.003). In T2DM group, significantly higher HDL-C  and HOMA-IR  were found in sub-group with higher vaspin levels  was found in controls. In T2DM group, vaspin was significantly positively correlated with BMI , WHR  and HOMA-IR . All results of Pearson correlation analysis were shown in Significant positive correlation between vaspin and HDL-C .P = 0.021). Similarly, higher percentage of new onset of T2DM was also found in sub-group with low adiponectin levels, compared with sub-group with high adiponectin levels. However, the statistical analysis was not significant . It was found that higher FPG and BMI, lower serum vaspin and adiponectin levels at baseline were independent risk factors for the occurrence of T2DM during 2.0-year follow-up. Results of multiple stepwise logistic regression analysis were shown in P = 0.015) and 0.35 , respectively.Among 170 subjects without T2DM at baseline, 11 developed T2DM during follow-up. 3 and 6 subjects were diagnosed with impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) respectively. As were shown in P≤0.05). At follow-up, FPG , 2h-PG  and HbA1c  were all significantly lower in sub-group with high vaspin levels than those in sub-group with low vaspin levels. In T2DM group, HbA1c was significantly lowered in sub-groups with either low or high vaspin levels at follow-up than baseline (both P = 0.008). There was no significant difference of the glycemic control between sub-groups with low and high vaspin levels at follow-up. . Compared with sub-group with low adiponectin level, sub-group with high adiponectin level had less patients receiving insulin treatment both at baseline  and follow-up .As were shown in Vaspin is a novel adipocytokine that is supposed to have insulin sensitizing effects Although results of animal studies indicate that vaspin is an insulin-sensitizing adipocytokine, the real role of vaspin in human insulin resistance is still unclear Gulcelik's research found that serum vaspin levels in diabetic patients with chronic complications including neuropathy, retinopathy and nephropathy were lower than those without above complications In our study, it was found that adiponectin was an independent risk factor for new onset of T2DM in non-diabetic subjects at follow-up, which was accordant with some other studies Several limitations should be considered before results interpretation. First, the control subjects were not normal people, but inpatients with hypertension or other mild cardiovascular diseases. This inclusion method can not exclude some confounding effects, including the effects of diseases itself and drugs treatment. However, people with high risk of T2DM constituted the control group, which might be partially responsible for the high occurrence of T2DM at follow-up and higher statistical power in small sample size. Second, the T2DM patients were not enrolled at the onset of the disease. The different duration of T2DM and different treatment in each patient may incorporate a possible source of selection bias. Third, the relatively small sample size in this study will decrease the statistical power, and further studies with larger sample size should be performed in the future to validate our results. Fourth, the relatively short follow-up period will weaken the effects of vaspin on glucose levels and chronic complications in T2DM group. Nevertheless, this cohort study provides support to the role of vaspin in glycemic control in non-diabetic people.In conclusion, low circulating vaspin level, as well as adiponectin level can be used as risk factors for the progression of T2DM. Further prospective observational studies are required to explain how a decrease in circulating vaspin level may be involved in the progression of T2DM.Table S1Basic clinical characteristics of all subjects at baseline. Note: BMI: body mass index; WHR: waist-hip ratio; SBP: systolic blood pressure; DBP: diastolic blood pressure; BUN: blood urea nitrogen; TC: total cholesterol; TG: triglycerides; HDL-C: high-density lipoprotein cholesterol; LDL-C: low density lipoprotein-cholesterol; FPG: fasting plasma glucose; 2h-PG: 2-h postprandial plasma glucose; HbA1c: glycosylated haemoglobin A1c; HOMA-IR: homeostasis model assessment of insulin resistance;*logarithmically transformed before analysis. Adjusted P values were from covariance analysis by adjusting for age, sex and BMI.(DOC)Click here for additional data file.


A Chinese human enterovirus 85 (HEV85) isolate, HTYT-ARL-AFP02F/XJ/CHN/2011, was isolated from a stool specimen of a child with acute flaccid paralysis in Xinjiang, China, in 2011. The complete genome sequence revealed that a natural intertypic recombination event had occurred between HEV85 and a previously undescribed serotype of HEV-B. Enterovirus within the family Picornaviridae, order Picornavirales, and include four species: HEV-A, HEV-B, HEV-C, and HEV-D (Human enteroviruses (HEVs) are members of the genus nd HEV-D . HEV85 bnd HEV-D –5.The prototype strain of HEV85, strain BAN00-10353/BAN/2000, was identified in a stool sample from a patient who had presented with AFP in Bangladesh in 2000 . At presThe complete genome sequence of the Chinese HEV85 isolate was acquired according to the published strategies for HEV sequencing after purification by plaque assay –9. Raw sP1 coding region, but it did not show high sequence homology with the P2 and P3 coding regions. Furthermore, the high bootstrap value obtained in Bootscan analyses strongly suggests that recombination events occurred between HEV85 and a new serotype HEV-B that has not been previously described. The likely crossover site appears to be after nt 3370 in the 2A region. These findings highlight that recombination is a common phenomenon occurring in HEVs  long and is composed of a single, large open reading frame of 6,579 nt that encodes a polyprotein of 2,191 amino acids. The nucleotide and amino acid sequence similarities between the Chinese HEV85 strain and the prototype strain were 86.66% and 98.08%, respectively. Phylogenetic analysis showed that they were clustered with the HEV85 prototype strain for the  in HEVs –15 and tTogether with the isolation of HEV85, a novel and recently identified HEV serotype, the trapping of an unknown new serotype HEV-B donor sequence in the Chinese HEV85 recombinant described in this study suggests that new HEV-B serotypes may be in circulation in the Xinjiang region of China. Hence, we have increased HEV surveillance in the Xinjiang region to determine the precise donor sequence of the new serotype HEV.JX898909.The nucleotide sequence of the complete genome of HEV85 recombinant strain HTYT-ARL-AFP02F/XJ/CHN/2011 has been submitted to GenBank under accession number


Origanummajorana exhibits anticancer activity by promoting cell cycle arrest and apoptosis of the metastatic MDA-MB-231 breast cancer cell line. Here, we extended our study by investigating the effect of Omajorana.  on the migration, invasion and tumor growth of these cells.We have recently reported that Omajorana.  significantly inhibited the migration and invasion of the MDA-MB-231 cells as shown by wound-healing and matrigel invasion assays. We also show that Omajorana.  induce homotypic aggregation of MDA-MB-231 associated with an upregulation of E-cadherin protein and promoter activity. Furthermore, we show that Omajorana.  decrease the adhesion of MDA-MB-231 to HUVECs and inhibits transendothelial migration of MDA-MB-231 through TNF-α-activated HUVECs. Gelatin zymography assay shows that Omajorana.  suppresses the activities of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). ELISA, RT-PCR and Western blot results revealed that Omajorana.  decreases the expression of MMP-2, MMP-9, urokinase plasminogen activator receptor (uPAR), ICAM-1 and VEGF. Further investigation revealed that Omajorana.  suppresses the phosphorylation of IκB, downregulates the nuclear level of NFκB and reduces Nitric Oxide (NO) production in MDA-MB-231 cells. Most importantly, by using chick embryo tumor growth assay, we also show that Omajorana.  promotes inhibition of tumor growth and metastasis in vivo.We demonstrate that non-cytotoxic concentrations of Origanummajorana as a promising chemopreventive and therapeutic candidate that modulate breast cancer growth and metastasis.Our findings identify  Breast cancer is the leading cause of cancer-related deaths in women worldwide. Approximately one-third of all women with breast cancer develops metastasis and ultimately dies as a result of the effects of the disease ,2. CanceAdhering of tumor cells to endothelial cells is an essential step during cancer progression and metastasis. Several adhesive molecules, such as intracellular adhesion molecule-1 (ICAM-1), have been identified as being responsible for the endothelial adhesion of cancer cells . While IAngiogenesis is a complex multistep process involving soluble factors, adhesion molecules, proteases and cytokines. The process of tumor angiogenesis starts when tumor cells themselves secrete and activate angiogenic factors, thereby activating proteolytic enzymes. At this time, endothelial cells concurrently proliferate, migrate, and differentiate. Vascular endothelial growth factor (VEGF) is the most prominent mediator in tumor angiogenesis that is markedly induced in breast cancer . Up-reguDegradation of the extracellular matrix (ECM) surrounding the primary tumor is an essential step in cancer cells invasion. This degradation is important for tissue remodeling and induction of angiogenesis, and is mainly mediated by specific proteolytic enzymes systems mainly matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Among all MMPs, upregulation of MMP-2 and MMP-9 was shown to be associated with breast cancer metastasis and poor clinical outcome . NortherMMPs are directly activated by the serine protease plasmin, which is activated from its proenzyme form (plasminogen) by the serine protease urokinase-type plasminogen activator (uPA) upon binding to cell surface receptor (uPAR). Overexpression of uPA has been found in many tumor types and is correlated with poor prognosis. Moreover, binding of uPA to uPAR also induces signal transduction that allows enhanced cell migration ,15–17. TNuclear factor κB (NFκB), a transcription factor, is a key player in cancer metastasis . It has Glucidum.  [Phytochemicals have accumulated increasing importance with regards to their ability to decrease various tumors growth, such as those in the breast. In regards to breast cancer, increasing data have shown that these phytochemicals modulate multiple pathways used by cancer cells in the processes of cell proliferation, survival, angiogenesis, invasion, and metastasis . Disruptlucidum .Origanummajorana, commonly known as marjoram, is spread worldwide. It is utilized as a spice and flavoring agent, and in traditional medicine as well for the treatment of chest infection, cough, sore throat, rheumatic pain, nervous disorders, stomach disorders, cardiovascular diseases, and skin care. There is increasing evidence that Omajorana.  possesses extensive range of biological activity, including antioxidant, antimicrobial, anti-inflammatory and hepatoprotective activities [Origanummajorana suppresses the growth of the triple negative MDA-MB-231 breast cancer cells by causing cell cycle arrest and apoptosis [Omajorana.  to inhibit migration, invasion and metastasis of the MDA-MB-231 cells.tivities –30. ReceOriganummarjorana ethanolic extract (OME) was prepared as previously described [escribed .Human breast cancer cells MDA-MB-231 and MDA-MB-231-GFP were maintained in DMEM . MDA-MB-23-GFP used in this study were previously described . All medWound healing assay was performed as previously described . Briefly4 cells were seeded in the upper chamber, HTS multiwell insert system , in serum-free medium, with or without OME and migration assay was carried out according to the manufacturer’s instruction. Serum-containing medium was added to the lower chamber to act as a chemotactic attractant. After 6 hours of incubation, cells were washed with PBS, fixed with formaldehyde. Cells were then stained with 1% crystal violet for 10 minutes. After washing with PBS, cells from at least 5 different random fields were counted under a microscope.5 x 10Cells were seeded in triplicate in 96-well plates at a density of 5,000 cells / well. After 24h of culture, cells treated with or without OME and incubated for the indicated time period. Cell viability was determined using a CellTiter-Glo Luminescent Cell Viability assay , based on quantification of ATP, which signals the presence of metabolically active cells. Luminescent signal was measured using Berthold FB12 Luminometer. Data were presented as proportional viability (%) by comparing the treated group with the untreated cells, the viability of which is assumed to be 100%.6 cells/ml was aliquoted into microcentrifuge tubes, washed with PBS, resuspended in 1m culture media and incubated with or without OME on a rocker for 1 h at 37°C. Cells were then fixed with 1% formaldehyde and pictures taken under an inverted microscope (Olympus IX71). Aggregation was calculated as previously reported [Growing cells were detached using 2mM EDTA in calcium magnesium-free PBS (CMF-PBS). A cell suspension of 1 x 10reported  using thRenilla luciferase were resuspended in HBSS containing 1% BSA at a concentration of 2x105 cells/ml. 150 µl of this cell suspension was added to the upper chamber containing confluent HUVECs in the absence or presence of OME. After 1 hour, unattached cells were removed by gently washing the plates three times with PBS. Adherent cells were lysed using a luciferase lysis buffer  and light units measured according to the manufacturer instructions.Adhesion of MDA-MB-231 to HUVECs was performed as previously described  with min5) cells placed in 0.5 mL of media containing vehicle or the indicated concentrations of OME were seeded into the upper chambers of the system; the bottom wells in the system were filled with DMEM complemented with 10% foetal bovine serum as a chemo-attractant and then incubated at 37°C for 24h. Non-penetrating cells were removed from the upper surface of the filter with a cotton swab. Cells that have migrated through the matrigel were fixed with 4% formaldehyde, stained with DAPI and counted in 6 random fields under a microscope. For quantification, the assay was done in duplicates and repeated three times.Invasion assay was performed as previously described . The invCells were seeded in 6-well plates in the absence of vehicle or OME for 24 hours. The conditioned medium was collected and the levels of secreted MMP-2 and MMP-9 were determined using immunoassay kits  according to the manufacturer’s protocol. Experiments were repeated three times and the average of three means is represented ± SEM.6) cells were incubated in serum-free DMEM for 24 h in the presence of vehicle or OME (150 and 300 µg/mL). The conditioned medium was collected, concentrated and 30 µg of total protein was resolved in 10% polyacrylamide gels containing 0.1% gelatin. After electrophoresis, the gels were washed for 1 h in 2.5% (v/v) Triton X-100 to remove SDS and then incubated overnight at 37 °C in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM ZnCl2 and 10 mM CaCl2 to allow proteolysis of the gelatin substrate. Bands corresponding to activity were visualized by negative staining using 0.5% Coomassie brilliant blue R-250 . Representative results from two independent experiments are shown. Densitometry was performed using ImageJ software and band density was normalized to the non-specific band staining on the gel.Gelatin zymography was performed as previously described . Briefly4) were grown in complete media on 4 well labtek chamber slide  for 24 h, then treated with ethanol as control or OME extract for 24 h at the indicated concentrations. Cells were then fixed in 10% formalin solution   for 5 min at RT followed by permeabilization in PBS containing 0.1% Triton X-100 for 5 min at RT. Cells were then washed three times with PBS, blocked with 5% nonfat dry milk in PBS for 30 min at RT and incubated with the primary antibody diluted, at the concentration suggested by the manufacturer, in 1% nonfat dry milk/PBS overnight at 4°C. Following overnight incubation, cells were washed three times with PBS and placed for 1 h at RT in the presence of fluorescein-conjugated secondary antibody diluted at 1:200 in 1% nonfat dry milk/PBS. After washing with PBS, cells were mounted in Fluoroschield with DAPI  and examined under Nikon Ti U fluorescence microscope.immunofluoreacence staining was performed as previously described . MDA-MB-5) were seeded in 24-well plates. The conditioned medium was collected and the level of VEGF therein measured using a VEGF enzyme-linked immunosorbent assay kit  according to the manufacturer’s protocol. Assays were performed in triplicates and three independent experiments were performed. Data are presented as mean values ± SEM.MDA-MB-231 cells , which is based on the Griess reaction, according to the manufacturer’s instructions. The value of nitrate/nitrite presented is the total value measured in the presence of cells minus the value determined from the media alone in the absence of any growing cells.Renilla luciferase reporter was used as an internal control, to which firefly luciferase values were normalized. Experiments were repeated three times and the average of three means is represented ± SEM.MDA-MB-231 cells were seeded in 12-well plates the day before transfection. Cells were then transfected with the E-cadherin luciferase reporter expression plasmid  using Li5/well) were then seeded onto the rehydrated membrane and allowed to grow until a confluent monolayer is formed. Where mentioned, TNF-α (25ng/ml) was added for 6 hours to stimulate HUVECs. Then, MDAMB 231 cells (1 x 106) were then loaded on top and incubated overnight in the absence or presence of OME. Cells on the upper chamber were removed with a cotton swab, whereas MDA-MB-231 on the bottom were stained and quantified as previously reported [Transendothelial migration of MDA-MB-231 through HUVEC was performed as previously described . Brieflyreported .5′-TCTCCTGACATTGACCTTGGC -3′, and antisense: 5′-CAAGGTGCTGGCTGAGTAGATC -3′; MMP9 sense, 5′- TTGACAGCGACAAGAAGTGG-3′, and antisense, 5′- CCCTCAGTGAAGCGGTACAT-3′; GAPDH sense, 5′-GGCCTCCAAGGAGTAAGACC -3′, and antisense: 5′- AGGGGTCTACATGGCAACTG-3′. The PCR products were separated by 1.5% agarose gel and visualized by ethidium bromide staining. Representative results from two independent experiments are shown.Total RNA from vehicle- or OME-treated MDA-MB-231 cells were prepared using Trizol reagent as described by the manufacturer . RNA expression of MMP-2 and MMP-9 was determined by RT-PCR. RT-PCR was performed using the Qiagen OneStep RT-PCR kit  according to manufacturer’s instruction. Equal amounts of RNA (500 ng) were used as templates in each reaction. The sequences of specific primers were as follows: MMP-2 sense, 6) were seeded in 100mm culture dishes and cultured for 24h before addition of various concentrations of OME extract. For whole cell lysates, after incubation for the indicated times, cells were washed twice with ice-cold PBS, released by scrapping, pelleted and lysed in RIPA buffer supplemented with protease/phosphatase inhibitor cocktail. Following incubation for 30 min on ice, the cell lysate was obtained by centrifugation at 14,000 rpm for 20 min at 4°C. Nuclear extract were prepared from vehicle- or OME-treated MDA-MB-231 cells using the NE-PER extraction reagents  according to the manufacturer’s instructions. Protein concentration of lysates was determined by BCA protein assay kit  and the lysates were adjusted with lysis buffer. Aliquots of 30 µg of total proteins were resolved onto 10-12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes  and blocked for 1 h at room temperature with 5% non-fat dry milk in TBST (TBS and 0.05% Tween 20). Incubation with specific primary antibodies was performed in blocking buffer overnight at 4°C. Horseradish peroxidise-conjugated anti-IgG was used as secondary antibody. Immunoreactive bands were detected by ECL chemiluminescent substrate . Membrane stripping by incubating the membrane in Restore western blot stripping buffer  according to the manufacturer’s instructions.Cells . One day later, tumors that began to be detectable were treated every second day at E11, E13, E15 and E17 by dropping 100 µl of either OME (300 µg/mL or 450 µg/mL), colchicine (2µM) or 0.02% ethanol (vehicle) in PBS onto the tumor. At E19 the upper portion of the CAM was removed, transferred in PBS and the tumors were then carefully cut away from normal CAM tissue and weighted. In parallel, a 1cm2 portion of the lower CAM was collected to evaluate the number of nodules, containing GFP-expressing cells. The fluorescent nodule were visualized in situ using whole mounts of tissue fixed in 4% formaldehyde in PBS and flattened between a hollow glass slide and a thick coverslip. In order to number the nodule, a thorough and complete visual scan of the piece of the lower CAM was done using fluorescent microscope. Chick embryos were sacrificed by decapitation.The chick embryo tumor growth assay was performed as previously described  with slit-test for paired or unpaired values was performed and a p value of < 0.05 was considered statistically significant.Results were expressed as means ± S.E.M. of the number of experiments. A Student’s We have recently reported that OME induces cell cycle arrest and apoptosis in MDA-MB-231 cells [Lost capacity for homotypic adherence is also associated with cancer cell metastasis. To determine whether OME would affect the cell-cell adherence behaviour of MDA-MB 231, cell aggregation assay was performed on vehicle and OME-treated cells. We found that OME significantly increased the ability of MDA-MB-231 cells to form cell aggregates visible as early as 30 min post-treatment  -2 and -9, among other MMPs, are known to play an important role in breast cancer cell invasion and metastasis. To test whether Next, we decided to examine the effect of OME on the activity of MMP-2 and MMP-9. MDA-MB-231 cells were treated with 150 and 300 µg/mL OME for 24 h in serum free DMEM, the media was collected, concentrated and tested for MMP2 and MMP-9 activity by gelatin zymography. As it is shown in The urokinase plasminogen activator (uPA) and its receptor (uPAR) were also shown to play a crucial role in breast cancer cell invasion and metastasis. Therefore, we examined whether OME also alters the expression of uPAR. Western blot analysis clearly shows that the expression level of uPAR was markedly decreased in OME-treated MDA-MB-231 cells -1, has been shown to play an important role in the adhesion of cancer cells to endothelial cells and therefore in metastasis, we sought to examine whether OME affects the expression of this adhesion molecule in MDA-MB-231 cells. Toward this aim, cells were treated with various concentrations of OME and the protein level of the ICAM-1 was determined by Western blotting. As it is shown in Since the migration of tumor cells through the vascular endothelium is another crucial event in the metastasis process, we used the transendothelial migration assay to investigate the effect of OME on the ability of MDA-MB-231 cells to migrate across a monolayer of endothelial (HUVEC) cells. As seen in Tumor growth and metastasis critically depend on angiogenesis. VEGF, a pro-angiogenic growth factor, has been shown to play an important role in this process. Thus, we investigated the effect of OME on the production of VEGF by both the MDA-MB-231 and endothelial cells (HUVEC) without or with the presence of TNF-α. We first examined the effect of OME of basal expression level of VEGF in MDA-MB-231 cells. Cells we grown with various concentrations of OME and the conditioned medium were collected and the level of VEGF measured by ELISA. Data shown on The NFκB signaling pathway is known to regulate the expression of various genes involved in tumor cells invasion. To investigate the effect of OME on the activation of NFκB signaling pathway, we first examined, by Western blotting, the phosphorylation status of IκB in OME-treated MDA-MB-231 cells. We found that OME drastically inhibited the phosphorylation of IκB (Nitric oxide (NO) signaling has also been shown to promote breast tumor growth and metastasis by altering the expression of genes implicated in cellular migration, invasion and angiogenesis –41. To tin vitro anti-breast cancer activities of Omajorana. , we decided to investigate its effect on tumor growth in vivo by using the chick embryo model. MDA-MB-231 cells were grafted on the chorioallantoic membrane (CAM) and formed tumors were treated every 48 h with vehicle, colchicine (2 µM) or increased concentrations of OME (300 and 450 µg/mL). At E 19, tumors were recovered from the upper CAM and weighted. As it is shown in To further confirm the in vivo.We next assessed for the ability of OME to inhibit metastasis by counting the number of nodules in the lower CAM in vehicle, colchicines and OME treated tumors. An average of 6.6 nodules were counted in the lower CAM of vehicle-treated chick embryo, while an average of 1.1 nodules only were counted in 300 and 450 µg/mL OME-treated embryo  production, both signalings involved in cancer cell invasion. Moreover, we demonstrated that Omajorana.  significantly inhibited tumor growth and metastasis in vivo in chick tumor growth assay.Tumor invasion and metastasis is a multistep process involving cell adhesion, proteolytic degradation and migration through the ECM and angiogenesis. Common cancer treatment drugs aim at blocking cell cycle progression, inducing cell death and/or inhibiting tumor migration and invasion. Cancer chemoprevention through these events has been reported for several natural compounds ,17,42–44Omajorana.  inhibited cell migration and promoted homotypic cell-cell aggregation and induced overexpression of E-cadherin in the MDA-MB-231 cells. We postulate that Omajorana.  exerts its anti-migratory effect on breast cancer cells, at least partly, through reactivation of the expression of E-cadherin gene. In fact we showed that OME was able to induce transactivation of E-cadherin promoter in transfected cells  involved in this regulation. Further investigation will be needed to decipher the exact mechanism by which Omajorana.  exert its effect on E-cadherin expression.It is well known that disruption of cell-cell adhesion during cancer progression is the initial stage required for the acquisition of invasive properties. Decreased cell-cell adhesion in cancer cells is often characterized by diminished expression of E-cadherin. Indeed, E-cadherin, a calcium-dependent, cell adhesion molecule, is considered as a tumor suppressor in breast cancer . A decrecadherin ,49. Histluteolin , a dietaluteolin . Based oOmajorana.  exerts its anti-invasive affect against MDA-MB-231 cells by significantly downregulating the expression of uPAR, MMP-2 and MMP-9 and decreasing the activity of these two proteases and consequently reducing ECM degradation.The dissemination of cancer cells from the primary tumor is another crucial event in the process of cancer invasion and metastasis, which involves the degradation of the ECM and the components of the basement membrane through proteases. Of these proteases, MMPs such as MMP-2, MMP-9 and the uPA are thought to play a key role in cancer cell invasion and metastasis ,52. It hThe ability of tumor cells to metastasize also largely depends on their ability to adhere and transmigrate though endothelial cells. Cancer cell-endothelial cell interaction is mediated by various adhesive molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin .Salviamiltiorrhiza, efficiently inhibited the adhesion of MDA-MB-231 cells to HUVECs by downregulation the expression of ICAM-1 and VCAM-1 [Omajorana.  was able to reduce the expression level of ICAM-1 protein in MDA-MB-231 cells and blocked their adhesion to the human vascular endothelial cells (HUVECs). Hence, we propose that downregulation of ICAM-1 expression could be one mechanism by which Omajorana.  blocks MDA-MB-231 cells capabilities to adhere to HUVECs and consequently preventing their metastasis.Studies have shown that the level of ICAM-1 protein expression on the cell surface positively correlated with metastatic potential of several breast cancer cell lines . Moreoved VCAM-1 . Recent d VCAM-1 . Based oOmajorana.  significantly reduced the production of VEGF in both HUVECs and MDA-MB-231 cells and thus suggesting that OME not only inhibit breast cancer cell invasion but could also block angiogenesis.Angiogenesis, a process by which new blood vessels forms, is crucial for tumor growth and metastasis. Blockade of angiogenesis can inhibit both tumor growth and metastasis . Thus inOmajorana.  inhibited the phosphorylation of IκB and reduced the protein level of nuclear NFκB and thus suggesting that Omajorana.  might negatively regulate the activity of NFκB possibly by affecting the activity of the IKK complex. It is noteworthy to mention that Omajorana.  significantly reduced the expression of several NFκB downstream target genes  involved in tumor metastasis. It appears then, that the inhibitory effect on NFκB could account in the anti-metastatic effects of Omajorana. .The NFκB signaling pathway is known to regulate the expression of various genes involved in the process of tumor metastasis. Elevated levels of NFκB are frequently detected in breast cancer cells. Studies showed that inhibition of NFκB activity could suppress metastasis in breast cancer cells. In fact, studies showed that inactivation of NFκB in MDA-MB-231 breast cancer cells inhibit the expression of many downstream target genes involved in tumor metastasis such as MMP-2 , MMP-9 ,60,61, VOmajorana.  efficiently reduced the level of NO production in the MDA-MB-231 in dose-dependent manner suggesting that Omajorana.  might exert its anti-metastatic effect, at least partly, by modulating the NO production in MDA-MB-231 cells. As such, NO production may be an important target for chemoprevention and therapy by Omajorana.  for the ER(-), mutant p53 MDA-MB-231 cells.Nitric Oxide (NO), synthesized by several nitric oxide synthases (NOSs), nNOS/NOS-1, iNOS/NOS2 and eNOS/NOS3 is a signaling molecule that regulates several physiological responses such as vasodilatation, cell migration, immune reactions and apoptosis . InteresOmajorana.  possess an anti-invasive and anti-metastatic effects against the highly proliferative and highly invasive human MDA-MB-231 breast cancer cell line by modulating the activity and/or the expression of proteins regulating the process of cellular migration, adhesion invasion and angiogenesis such as E-cadherin, ICAM-1, MMP-2, MMP-9, uPAR and VEGF at least partly through inhibition of the NFκB and NO signaling pathways. Our results also showed that Omajorana.  inhibited tumor growth and metastasis in an in vivo tumor growth assay. Thus, our current study along with our previous findings, identify Origanummajorana as a promising chemopreventive and therapeutic candidate that inhibits breast cancer growth and metastasis by modulating the expression and activities of several targets. Nowadays, there is a growing interest in combination therapy using multiple anticancer drugs affecting several targets/pathways, thus Omajorana.  certainly merits a lot of attention for further explorations to identify novel compounds for breast cancer.In summary, this study clearly demonstrated, for the first time, that